The state of oxidative stress is a balance between prooxidant and antioxidant mechanisms. Increased oxidative stress is associated with various diseases such as coronary heart disease, neurodegenerative diseases, arthritis, and cataract formation. Antioxidant mechanisms exist in the body such as antioxidant enzymes and other small molecular antioxidants that can protect against harmful effects of free radicals. Among these, glutathione (GSH) is major antioxidant in human tissues, particularly in erythrocytes. Glutathione is a tripeptide with a free thiol group. Under the effect of glutathione peroxidase, GSH can remove H.sub.2 O.sub.2 at a high rate and in the process itself becomes oxidized glutathione (GSSG). The GSSG must then be converted back to GSH by the enzyme glutathione reductase. When erythrocytes are exposed to the increased oxidative stress, the ratio of GSH/GSSG will decrease, particularly evident in erythrocytes, as a consequence of GSSG accumulation. Therefore, the measurement of the GSH/GSSG ratio provides a significant index to evaluate the state of oxidative stress.
Since Tietze (1) developed an enzymatic method for quantitative determination of amounts of total and oxidized glutathione, the estimation of total glutathione in human tissues have been very successful. The method employs Ellman's reagent (5,5'-dithiobis-2-nitrobenzoic acid), which reacts with GSH to form a spectrophotometrically detectable product. GSSG may be determined by reduction of GSSG to GSH with glutathione reductase and a reductant such as NADPH (.beta.-nicotinamide adenine dinucleotide phosphate, reduced form), and reaction of formed GSH (and any GSH in the original sample) with Ellman's reagent.
However, the accurate measurement of tissue GSSG levels has proved very difficult due to either the lower amount of this form in tissue, or because of the absence of effective methods to prevent oxidation of GSH to GSSG during the process of sample preparation. In order to measure the GSSG in tissue, Guntherberg and Rost (2) first introduced N-ethylmaleimide (NEM) to eliminate the GSH. Although NEM can react with GSH to form a stable complex and prevent the participation of the reduced form in the enzymatic assay, NEM also inhibits glutathione reductase. Therefore, NEM must be removed from the sample before enzymatic assay. For this reason, Griffith (3) first introduced 2-vinylpyridine (2-VP) to derivatize GSH, since 2-vinylpyridine does not inhibit glutathione reductase significantly. Although 2-VP can react with GSH, it is a slowly reactive reagent and has little solubility in aqueous medium. The reaction usually takes about 60 min to remove 70% GSH in the sample with 10 mM 2-VP, during which time oxidation of GSH may occur, obscuring the level of GSSG. Furthermore, 2-VP interferes with the glutathione reductase method to some extent. To date, many reports indicate that the GSSG level in normal blood is up to 50 mM, which might be significantly overestimated by oxidation of GSH in samples, particularly in a red blood cell lysate, to GSSG in the absence of optimal sample preparation procedures to prevent formation of GSSG.
French patent application Serial No. 91.14782 (PCT/FR92/01093; U.S. Pat. No. 5,543,298) identifies 1-methyl-2-vinylpyridinium trifluoromethanesulfonate (M2VP); 1,4,6-trimethyl-2-vinylpyridinium tetrafluoroborate (TM2VP); and 1-methyl-4-vinylquinolinium tetrafluoroborate (M4VP) as mercaptan scavenging compounds useful for assaying superoxide dismutase activity.
It is toward the development of a method to quantitate levels of GSSG by preventing oxidation of GSH in a sample and permitting the measurement of GSSG that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.